Project Summary/Abstract: Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcer disease. Humans are the only known natural host for H. ducreyi, and chancroid remains one of the least understood STIs. A large number of genes encoding putative virulence factors of this organism have been identified. However, testing in the human challenge model for experimental chancroid revealed that only a handful of these genes are truly essential for full virulence of H. ducreyi. Among these, inactivation of lspA1 and lspA2 resulted in the most severe attenuation of H. ducreyi. LspA1 and LspA2 are very large proteins (456 kDa and 543 kDa, respectively) with 86% identity; each protein can independently inhibit Fc? receptor-mediated phagocytosis. Our studies focus on LspA1, the smaller of these proteins. We determined that the minimum domain required for phagocytosis inhibition is a 63 amino acid (aa) segment of LspA1. Our new data indicate that LspA1 exhibits at least three additional and previously undescribed activities. These include nuclear localization in mammalian cells, autoproteolytic processing, and induction of cell rounding. One domain of LspA1 localizes to the eukaryotic nucleus and alters the mammalian transcriptome, affecting expression of genes involved in skin homeostasis and adaptive immunity. Experiments in the first Specific Aim will further define the effects of this nuclear localization on host transcriptional regulation and the ramifications of these transcriptional changes for chancroid pathogenesis. We also discovered that the YopT homology region in LspA1 has autoprocessing cysteine protease activity, resulting in the release of a 316-aa fragment designated the cleavage product (CP). CP causes mammalian cells to round, indicating that LspA1 can exert toxin-like effects. CP interacts, either directly or indirectly, with Rac1, a small Rho GTPase, which suggests that CP causes cell rounding by altering cytoskeletal signal transduction pathways. Determination of the molecular basis for this cell rounding activity constitutes the second Specific Aim. Studying the effectors in this multi-faceted protein will advance our knowledge not only about chancroid pathogenesis, but also about mammalian systems targeted by bacterial virulence factors.